HISTONE/DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

描述(改编自申请者摘要)：长期目标 本研究计划的目的是阐明体细胞分裂的机制。 染色质的生物合成，并了解表观遗传的繁殖 信息传给子代细胞。为此目的，本提案侧重于 组蛋白乙酰化和磷酸化在细胞周期中的作用 染色质复制，以及对核小体预组装的分析 在转化的人类细胞HeLa中观察到的复合体 排队。 将进行实验来解剖可溶的非 DNA复制时积累的核小体组蛋白复合体 抑制，并鉴定可能的体细胞组蛋白护送蛋白。 这将通过1)免疫沉淀放射性标记细胞来完成 含有抗组蛋白抗体的提取物，包括特异性抗体 乙酰化H4；和2)用抗体探测纯化的络合物 识别已知的体细胞核小体组装因子。这个 H4乙酰化参与护航蛋白识别也将 通过用人工合成的方法挑战体细胞提取液进行评估 代表乙酰化或非乙酰化的多肽 组蛋白H4的N-末端。在平行实验中， 可溶性组蛋白复合体参与体内核小体组装 而体外试验也将被确定。 作为上述研究的一部分，一种胞浆组蛋白的性质 乙酰转移酶，初步实验强烈表明 参与新合成的组蛋白H4的乙酰化，将是 检查过了。它的活性、特异性和细胞周期调节 乙酰转移酶(专用于H4，似乎是 “乙酰转移酶B”家族)将通过 简单的体外实验，使用纯化的组蛋白或 以乙酰化和非乙酰化的H4N端肽为底物。这个 这种乙酰转移酶促进组蛋白复合体的能力 编队也将被检查。 最后，识别磷酸化的抗体，但不识别 组蛋白H1的非磷酸化形式将被用来研究 染色质复制中的H1磷酸化。免疫共沉淀法 将对交联型(放射性标记)染色质片段进行 确定磷酸化的H1相对于新的 复制的DNA和新组装的核小体。通过 免疫沉淀法复制过程中标记的染色质DNA 环己酰亚胺，磷酸化H1的相对程度 还将对分离的亲本核小体进行分析。

DESCRIPTION (Adapted from Applicant's Abstract): The long-term objective of this research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the propagation of epigenetic information to progeny cells. To this end, the present proposal focuses on the function of histone acetylation and phosphorylation during chromatin replication, and on the analysis of nucleosome pre-assembly complexes that have been observed in HeLa, a transformed human cell line. Experiments will be performed to dissect the composition of soluble non- nucleosomal histone complexes that accumulate when DNA replication is inhibited, and to identify possible somatic histone escort proteins. This will be accomplished by 1) immunoprecipitating radiolabeled cell extracts with anti-histone antibodies, including antibodies specific for acetylated H4; and 2) probing the purified complexes with antibodies that recognize known somatic nucleosome assembly factors. The involvement of H4 acetylation in escort protein recognition will also be assessed, by challenging somatic cell extract with synthetic polypeptides that represent either the acetylated or the unacetylated N-terminus of histone H4. In parallel experiments, the ability of soluble histone complexes to participate in nucleosome assembly in vivo and in vitro will also be determined. As part of the above studies, the properties of a cytosolic histone acetyltransferase, which preliminary experiments strongly suggest is involved in acetylation of newly synthesized histone H4, will be examined. The activity, specificity, and cell cycle regulation of this acetyltransferase (which is specific for H4, and appears to be a member of the "acetyltransferase B" family) will be determined through a straightforward in vitro assay, using either purified histones or acetylated and unacetylated H4 N-terminal peptides as substrates. The ability of this acetyltransferase to facilitate histone complex formation will also be examined. Finally, antibodies that recognize the phosphorylated, but not the unphosphorylated forms of histone H1 will be used to study the role of H1 phosphorylation in chromatin replication. Immunoprecipitations of cross- linked (radiolabeled) chromatin fragments will be performed to determine the distribution of phosphorylated H1 with respect to newly replicated DNA, and to newly assembled nucleosomes. By immunoprecipitating chromatin DNA labeled during replication in cycloheximide, the relative degree to which phosphorylated H1 is presented on segregated parental nucleosomes will also be analyzed.